Universal nucleotides for nucleic acid analysis
View Patent ↗The invention includes methods and kits for making and analyzing primer extension products incorporating one or more universal bases, including methods and kits for nucleic acid sequencing and microsatellite analysis.
1. A kit for sequencing a target nucleic acid template, comprising at least one universal nucleotide, at least one polymerase, and at least one specific terminator; wherein one or more of the at least one universal nucleotides is individually selected from 2′-deoxy-7-azaindole-5′-triphosphate (d7AITP), 2′-deoxy-isocarbostyril-5′-triphosphate (dICSTP), 2′-deoxy-propynylisocarbostyril-5′-triphosphate (dPICSTP), 2′-deoxy-6-methyl-7-azaindole-5′-triphosphate (dM7AITP), 2′-deoxy-imidizopyridine-5′-triphosphate (dImPyTp), 2′-deoxy-pyrrollpyrizine-5′-triphosphate (dPPTP), 2′-deoxy-allenyl-7-azaindole-5′-triphosphate (dA7AITP), and 2′-deoxy-propynyl-7-azaindole-5′-triphosphate (dP7AITP).
2. The kit of claim 1 , wherein one or more of the at least one specific terminators comprises a label.
3. The kit of claim 2 , wherein the label comprises a fluorescent dye.
4. The kit of claim 3 , wherein the label comprises an energy-transfer fluorescent dye.
5. The kit of claim 1 , further comprising at least one primer.
6. A kit for detecting a short tandem repeat in a target nucleic acid template, comprising at least one universal nucleotide, at least one polymerase, and at least one primer comprising a sequence that is complementary to a sequence adjacent to a short tandem repeat in the target nucleic acid template; wherein one or more of the at least one universal nucleotides is individually selected from 2′-deoxy-7-azaindole-5′-triphosphate (d7AITP), 2′-deoxy-isocarbostyril-5′-triphosphate (dICSTP), 2′-deoxy-propynylisocarbostyril-5′-triphosphate (dPICSTP), 2′-deoxy-6-methyl-7-azaindole-5′-triphosphate (dM7AITP), 2′-deoxy-imidizopyridine-5′-triphosphate (dImPyTp), 2′-deoxy-pyrrollpyrizine-5′-triphosphate (dPPTP), 2′-deoxy-allenyl-7-azaindole-5′-triphosphate (dA7AITP), and 2′-deoxy-propynyl-7-azaindole-5′-triphosphate (dP7AITP).
7. The kit of claim 6 , wherein one or more of the at least one primers comprises a label.
8. The kit of claim 7 , wherein the label comprises a fluorescent dye.
9. The kit of claim 8 , wherein the label comprises an energy-transfer fluorescent dye.
10. The kit of claim 6 , wherein the at least one primer is two primers.